Mutations in voltage-dependent sodium channel genes cause neurological, muscular, and cardiac diseases. Although much is known about sodium channel function, there is still much to learn. The sodium channel is a large molecule and understanding which of its many amino acids is important to its function is a daunting task. One method to pinpoint potentially important amino acids in a protein is to align the sequences of the same protein from a number of different organisms to find out which amino acids do NOT change over the course of evolution. Another lesser-used strategy that we employ here is to include sequences from organisms in which that molecule is under strong selection pressure to evolve. In this case, we look for amino acid changes that DO change over the course of evolution. Weakly electric fish generate electric organ discharges (EODs) for communication and sensing objects. EODs are generated by sodium channels and the sodium channels of electric fish have undergone strong selection for species-specific changes in amino acids in critical regions of the channel. Using the approach described above, we have already discovered a novel functional domain of sodium channels. We will continue to study the process of evolution of sodium channel genes in the two independently evolved groups of electric fish in order to discover other functionally important regions of the channel. Using bioinformatics approaches to compare amino acid and nucleotide sequences in the two groups of electric fish, non-electric fish, and other vertebrates, including humans, we will detect amino acids sites that are likely under positive selection in the electric fish's sodium channels. We will then test whether these particular amino acids are truly important to channel function by introducing the amino acid changes that we observe in electric fish sodium channels into a human muscle sodium channel (as well as the converse) and determining whether these alter the biophysical properties of expressed sodium channels. Besides providing insights into the evolutionary processes, this work will aid our basic understanding of the functioning of sodium channels, a clinically important family of ion channels. Project Narrative: Mutations in sodium channel genes cause neurological, muscular, and cardiac diseases. Using bioinformatics approaches we will identify amino acids that are likely to be important to the function of the sodium channel. We will then test whether these particular amino acids are truly important to channel function by perturbing these amino acid in a human muscle sodium channel gene and observing whether these alter the biophysical properties of the sodium channels. This work will be medically important to understand the function of sodium channels, a clinically important family of ion channels.